Krause Kirsten, Lopes de Souza Renata, Roberts Douglas G W, Dieckmann Carol L
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA.
Mol Biol Cell. 2004 Jun;15(6):2674-83. doi: 10.1091/mbc.e04-02-0126. Epub 2004 Mar 26.
In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5' untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used. However, the protein is easily solubilized by omitting salt from the extraction buffer, which suggests Cbp1 is loosely associated with the membrane through weak hydrophobic interactions. Gel filtration analysis and blue native PAGE showed that Cbp1 is part of a single 900,000-Da complex. The complex was purified using the biotin tag and a sequence-specific protease cleavage site. In addition to Cbp1, the complex contains several polypeptides of molecular weights between 113 and 40 kDa. Among these, we identified another message-specific factor, Pet309, which promotes the stability and translation of mitochondrial cytochrome oxidase subunit I mRNA. A hypothesis is presented in which the Cbp1-Pet309 complex contains several message-specific RNA binding proteins and links transcription to translation of the mRNAs at the membrane.
在酿酒酵母中，核编码蛋白Cbp1通过与5'非翻译区相互作用促进线粒体细胞色素b转录本的稳定性和翻译。现在，将生物素结合肽标签融合到Cbp1的C末端后，可通过使用过氧化物酶偶联抗生物素蛋白在线粒体提取物中进行检测。当采用高离子强度提取条件时，Cbp1与线粒体膜相关联。然而，通过从提取缓冲液中省略盐，该蛋白很容易溶解，这表明Cbp1通过弱疏水相互作用与膜松散结合。凝胶过滤分析和蓝色天然聚丙烯酰胺凝胶电泳表明，Cbp1是一个单一的900,000道尔顿复合物的一部分。利用生物素标签和序列特异性蛋白酶切割位点对该复合物进行了纯化。除Cbp1外，该复合物还包含几种分子量在113至40 kDa之间的多肽。其中，我们鉴定出另一种信息特异性因子Pet309，它促进线粒体细胞色素氧化酶亚基I mRNA的稳定性和翻译。本文提出了一个假说，即Cbp1-Pet309复合物包含几种信息特异性RNA结合蛋白，并将转录与膜上mRNA的翻译联系起来。
